Hirudin is a small protein isolated from the salivary glands of the medicinal leech, Hirudo medicinalis. It is the most potent and most specific known inhibitor of thrombin, the serine protease that functions in regulating hemostasis and that catalyzes the final step in blood coagulation--the conversion of fibrinogen to clottable fibrin. Hirudin has been shown to be an effective anticoagulant and antithrombotic agent in animals and humans and may be uniquely suited to the clinical treatment of venous and arterial thrombosis--particularly as an adjunct to fibrinolytic therapy, disseminated intravascular coagulation, antithrombin-III deficiency, the control of fibrin deposition during wound healing, and some forms of metastatic cancer.
The purification and characterization of hirudin from the leech have been well-studied. The primary structures of three variants designated HV-1, HV-2, and HV-3 have been determined. Recently, Scharf et al., (1989) FEBS Lett 255:105 reported on ten additional hirudin sequences, strengthening the concept of hirudin as a "family of isoinhibitors."
Several laboratories have constructed synthetic genes for HV-1 and have expressed biologically active hirudin in microbial systems. These are reviewed in Johnson et al., (1989) Seminars in Thrombosis and Hemostasis 15(3):302. As shown in Table 1 therein, specific activities reported for purified recombinant hirudins show some variability, perhaps reflecting the degree of purity and/or differences in assay conditions. The naturally occurring protein is sulfated at tyrosine.sub.63, although purified preparations may contain as much as 12% of the nonsulfated form (Chang (1983) FEBS Lett 164:307). Desulfation of the tyrosine results in a three- to ten-fold decrease in affinity for thrombin (see, Stone and Hofsteenage (1986) Biochem 25:4622; Dodt et al., (1988) FEBS Lett 229:87) indicating that sulfation of tyrosine.sub.63 is responsible for the enhanced affinity of natural over recombinant hirudin.
Analogs of hirudin have also been produced by chemical modification or through recombinant DNA techniques. For example, European Patent Publication 273,800 discloses a hirudin analog having the putative native asparagine.sub.47 substituted with lysine, arginine or histidine and the native tyrosine.sub.63 substituted with glutamine or asparagine. As tested in a thrombin inhibition assay, the Arg.sub.47 and Lys.sub.47 variants of HV-2 are shown to be at least as active as native HV-2, while Glu.sub.63 or His.sub.47 variants are less active than the native form.
Degryse et al., (1989) Protein Engineering 2(6):459 similarly disclose that the Lys.sub.47 and Arg.sub.47 variants of HV-2 had lower (5- to 14-fold) dissociation constants than unmodified HV-2. (Actually, Lys.sub.47 is the native residue at position 47 of HV-2; Lys.sub.47 was designated an HV-2 variant due to a sequencing error.) Furthermore, the Lys.sub.47 protein displayed enhanced antithrombotic activity in vivo, having a 100-fold lower ED.sub.50 compared to HV-2 (Gln.sub.47) in the rabbit Wessler venous thrombosis model. These results demonstrate that in vivo antithrombotic efficacy correlates with the dissociation (inhibition) constant which is an in vitro quantitative measure of the inhibition of thrombin by hirudin.
U.S. Pat. No. 4,668,662 discloses chemically synthesized HV-1 analogs having amino acid substitutions in either of the first two amino-terminal positions, as well as a modified Tyr.sub.63. The hydroxyl position on the phenyl ring of Tyr63 may be a sulfate or a phosphate group. Hofsteenage et al., (1990) Eur J Biochem 188:55 incubated recombinant hirudin with [gamma-.sup.32 P] ATP and protein tyrosine kinase III to phosphorylate hirudin at Tyr.sub.63. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin.
U.S. Pat. No. 4,745,177 discloses desulfatohirudin and desulfatohirudin having the 2 carboxy-terminal residues removed. These proteins are produced by chemical or biological means to eliminate the sulfate ester from the hydroxyl group at Tyr.sub.63.
U.S. Pat. No. 4,767,742 discloses hirudin shortened at the amino-terminus by up to two amino acids and at the carboxy-terminus by up to 17 amino acids, as well as the desulfated derivatives.